Intensive studies have been carried out in the prior art on the breeding and improvement of strains of the genus Brevibacterium in order to achieve efficient production of amino acids. As a means for use in the breeding, a large number of genetic engineering techniques have been reported. Gene manipulation techniques for use in the breeding of strains belonging to the genus Brevibacterium have been developed making use of plasmid and phage systems, such as establishment of protoplast transformation methods (Katsumata, R., Ozaki, A., Oka, T. and Furuya, A.: J. Bacteriol. 159 (1984) 306-311, Santamaria, R. I., Gil, J. A. and Martin, J. F.: J. Bacteriol. 161 (1985) 463-467), development of various vectors (Miwa, K., Matsui, K. , Terabe, M., Nakamori, S., Sano, K. and Momose, H.: Agric. Biol. Chem. 48 (1984) 2901-2903, Katsumata, R., Ozaki, A., Oka, T. and Furuya, A.: J. Bacteriol. 159 (1984) 306-311, Santamaria, R., Gil, J. A., Mesas, J. M. and Martin, J. F.: J. Gen. Microbiol. 130 (1984) 2237-2246, Patek, M., Nesvera, J. and Hochmannova, J.: Appl. Microbiol. Biotechnol., 31 (1989) 65-69, Yeh, P., Oreglia, J., Prevotos, F. and Scicard, A. M.: Gene 47 (1986) 301-306), development of gene expression controlling method (Tsuchiya, M. and Morinaga, Y.: Bio/Technology 6 (1988) 428-430) and development of cosmids (Miwa, K. , Matsui, K. , Terabe, M., Ito, K., Ishida, M., Takagi, H., Nakamori, S. and Sano, K.: Gene 39 (1985) 281-286). Also reported are cloning of genes making use of such systems (Melumbres, M., Mateos, L. M. , Guerrero, C. and Martin, J. F.: Nucleic Acids Res., 16 (1988) 9859, Mateos, L. M., Del, R. G., Aguilar, A. and Martin, J. F.: Nucleic Acids Res., 15 (1987) 10598, Matsui, K., Miwa, K. and Sano, K.: Agric. Biol . Chem. 52 (1988) 525-531, Mateos, L. M., Del, R. G., Auilar, A. and Martin, J. F.: Nucleic Acids Res., 15 (1987) 3922, Matsui, K., Sano, K. and Ohtsubo, E.: Nucleic Acids Res., 14 (1986) 10113-10114, Peoples, O. P., Liebl, W., Bodis, M., Maeng, P. J., Follettie, M. T., Archer, J. A. and Shinskey, A. J.: Mol. Microbiol., 2 (1988) 63-72, Eikmanns, B. J., Follettie, M. T., Griot, M. U., Martin, U. and Shinskey, A. J.: Mol. Gen. Genet., 218 (1989) 330-339, O'Regan, M., Thierbach, G., Bachmann, B., VGilleval Lepage, P., Viret, J. F. and Lemoine, Y.: Gene 77 (1989) 237-251, Follettie, M. T. and Shinskey, A. J.: J. Bacteriol., 167 (1986) 695-702) and yield improvement of various amino acids (Sano, K., Ito, K., Miwa, K. and Nakamori, S.: Agric. Biol. Chem., 51 (1987) 597-599).
However, there are no reports on mobile genetic elements originated from strains belonging to the genus Brevibacterium.
Mobile genetic element is a DNA fragment which can move on the chromosome and its existence is known in a large variety of organisms ranging from procaryotic to eucaryotic organisms, especially in detail in corn, Drosophila, yeast and the like as eucaryotic organisms and Escherichia coli and the like as procaryotic organisms (Mobile DNA, American Society for Microbiology, Washington D.C. (1989)). Also, Japanese Patent Application Laying Open (Kokai) No. 63-24889 discloses a mobile genetic element originated from Corynebacterium diphtheriae and European Patent Application 0,445,385 A discloses a transposon originated from a bacterium belonging to the genus Corynebacterium.
Mobile genetic elements of bacterial origins are divided into insertion sequence and transposon. Insertion sequence is a DNA fragment having a size of from 760 bp to 2000 bp, with its both termini having inverted repeat sequences and its inner region encoding transposase which is an enzyme necessary for its moving. Transposon is a mobile genetic element having a gene which does not take part directly in the transfer, such as a drug resistant gene, and is present in a form in which said gene is interposed between two insertion sequences or inserted into an insertion sequence. As a characteristic feature common to insertion sequence and transposon, it is known that duplication of a nucleotide sequence of about 10 bp can be found in the periphery of a region where either of them is inserted (Mobile Genetic Elements, Academic Press, New York (1983) p159-221).
Some of the currently known mobile genetic elements, such as Escherichia coli transposons Tn 10 and Tn 5 and Mu phage, are markedly useful in genetic engineering. Examples of such use include gene disruption in which expression of a chromosomal gene is inhibited by inserting a transposon into the gene, gene expression in which a transposon having a promoter sequence inserted therein is transferred on a chromosome thereby effecting expression of its downstream sequence, and gene transfer in which a new gene is transferred into a chromosome by inserting the new gene into a transposon and effecting its transfer on the chromosome (Mobile DNA, American Society for Microbiology, Washington D.C. (1989) 879-925).
An object of the present invention is to obtain a DNA fragment containing a mobile genetic element originated from a strain belonging to the genus Brevibacterium, which is essential for the chromosome engineering-aided improvement of Brevibacterium strains that are important in the amino acid industry. Its another object is to provide a process for obtaining said mobile genetic element and establish a process for transferring a gene into chromosomal DNA making use of said mobile genetic element.